Plant stem cell product treatments

ABSTRACT

Embodiments of the invention include plant stem cell products and treatment devices for applying the plant stem cell products. In some embodiments, the products include extracts of plant stem cells, such as lingonberry stem cells and orchid stem cells, and an aerosolizing device configured to create an aerosol from the plant stem cell products and to deliver the aerosol to the lung via inhalation.

RELATED PATENT APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/388,802, filed Apr. 18, 2019, which claims the benefit of U.S.provisional patent application No. 62/776,237 entitled Aerosoltreatments, filed Dec. 6, 2018 by Maryam Rahimi. The entire content ofthese patent applications are incorporated by reference herein for allpurposes.

REFERENCE TO A SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The content of the ASCI text file of the sequence listing named“MR01C1_seq_ST25.txt”, which is 446 bytes in size was created on Mar.31, 2021 and electronically submitted via EFS-Web is incorporated hereinby reference in its entirety.

TECHNICAL FIELD

This invention relates to treatment of disease conditions by applicationof plant stem cell products. In embodiments, the invention includestreatment of lung conditions by inhalation of aerosols including plantstem cell products.

BACKGROUND

Plant extracts form the basis of most traditional medical treatments.Active materials isolated from plant extracts, ranging from aspirin tomorphine to paclitaxel to quinine form a large fraction of the modernpharmacopeia. The structural diversity of plant-derived compounds isenormous, and its exploration is still an active part of pharmaceuticaldevelopment. Plants are the source of numerous compounds of therapeuticvalue in human disease. The major classes of plant-based medicinesinclude alkaloids, glycosides, polyphenols and terpenes.

Multicellular plants, like animals, are composed of differentiatedtissues and stem cells. Plant stem cells are dedifferentiated cellscapable of division to create more stem cells and differentiated cellsor their precursors. These replicating cells may be isolated from a partof a plant regenerating from an injury or the progeny of such cells.Injury sites may be from any portion of a plant such as callus, leaves,fruit, stems, flowers, roots, meristem, root cap, or seeds. Other plantstem cells may be derived from cells of a developing plant embryo, froma plant callus, or from plant tissue samples (explants) in tissueculture medium in vitro. Plant stem cells may be derived from manydifferent cell types and may be able to differentiate into a wholeplant. Plant stem cells, as used in this document, includes at leastmeristem cells, callus-derived cells, injury-site derived cells, andembryonic cells. Meristem cells at ends of stalks and in the root apexprovide the plant with new cells and enable the plant to grow throughoutits life. A callus is a stage of somatic embryogenesis (i.e. zygoteformation without fertilization) followed by dedifferentiation producingstem cells capable of generating new plant tissue. The phrase plant stemcells as used herein includes extracts of such cells including productsof stem cell lysis or partial digestion.

Differentiated plant cells produce a wealth of compounds evolved tocombat predation, parasitism, disease, or adverse climatic conditions,to signal, or to encourage pollination or seed distribution.Differentiated plant cells such as roots, flowers, fruit, and seedsproduce many of these compounds. However, plant stem cells by theirnature lack the more specialized synthetic apparatus of differentiatedcells. Instead, plant stem cells would be expected to focus morenarrowly on producing materials needed to establish or replace othercell types, leaving the more diverse chemical synthesis tasks tospecialized progeny. For example, plant stem cells in sprouting seeds,such as the germinated barley used in brewing, direct much enzymaticactivity to producing simple sugars (such as the disaccharide maltose)to support the energy needs of a growing embryo.

Plant stem cells also have properties and functions that help stimulateand regenerate plants after injury and stress. This is most evident inthe recovery from physical injuries such as in a wound or callus.

As in other eukaryotes including mammals, plant cells include telomeresthat limit somatic cell division. Telomeres are ribonucleoproteinstructures capping the ends of chromosomes. Telomeres shorten with eachcell division, but telomerase, a reverse transcriptase, elongatestelomeres in stem cells. Telomerase expression profiles mirror changesin telomere length, with the highest enzyme levels and telomere lengthsassociated with cells that have unlimited capacity for proliferation.Cells with reduced length telomeres undergo senescence or apoptosis. Inlarge-bodied or long-lived animals, this process protects againstsomatic cell-derived cancers.

In vertebrates, telomeres are composed of 6-mer TTAGGG repeats coatedwith specific protecting proteins. Most plant telomeres (including modelorganism Arabidopsis thaliana) contain the 7-mer TTTAGGG that includesthe vertebrate 6-mer. The Arabidopsis-type telomere is found in mostangiosperms, but the sequence is absent in most monocot Asparagalesspecies. Many species within Asparagales include the 6-mer TTAGGGsequence found in humans. Apparently, this sequence was altered duringthe rise of the Asparagales, an order including more than ten percent ofall angiosperms. Onions (Allium cepa) are members of Asparagales andinclude the 12-mer telomere sequence CTCGGTTATGGG (SEQ ID NO:1), whichalso includes the human 6-mer sequence.

Human tissue may be afflicted with a variety of diseases and conditions,such as injury, wounds, infection, inflammation, infarct, cancer, ordiseases unique to specialized tissues. In some cases, these conditionsmay be treated by systemic administration of medications, includingplant-derived medications. In other cases, these conditions may betreated by local administration of therapeutic agents to avoid unwantedeffects outside the treated tissue or to limit the overall dosage.

Human lungs may be afflicted with a variety of disease or degenerativeconditions. These include, for example, cancers, bronchial constriction,asthma, chronic obstructive pulmonary disease, emphysema, cysticfibrosis, inflammatory conditions, infections, and other conditions.Some lung conditions may be treated by direct administration ofmedications to the lungs via aerosol inhalation. In some cases,plant-based materials may treat some of these conditions.

For example, plants of the genus Ephedra, including E. sinica andothers, produce the alkaloids ephedrine and pseudoephedrine in at leasttheir stems and leaves. However, when taken systemically, extracts ofEphedra species have been associated with adverse cardiovascular andrenal events and with anxiety, dizziness, difficulty urinating, drymouth, headache, irritation of the stomach, nausea, psychosis,restlessness, sleep problems, and tremors. The FDA banned the U.S. saleof dietary supplements containing ephedrine alkaloids, as posing anunreasonable risk of injury or illness.

Flavonoids are polyphenolic compounds (a subclass of flavanols) inplant-based foods. Quercetin, a strong antioxidant, is the major foodflavonoid. Quercetin can chelate metals, scavenge oxygen free radicalsand prevent the oxidation of low-density lipoprotein (LDL). Oxidized LDLis an intermediate in the formation of atherosclerotic plaques.Quercetin might therefore contribute to the prevention ofatherosclerosis: the intake of flavanols is inversely associated withsubsequent cardiovascular disease in several prospective epidemiologicalstudies. Epidemiological studies suggest that consumption of quercetinprotects against cardiovascular disease, but its absorption in man iscontroversial. Feeding studies of plant-based foods in humans foundquercetins from foods such as onions, which contain glucose conjugatesof quercetin, were more readily absorbed than non-conjugated quercetinsfrom apples or tea. Bioavailability of quercetin was about three timeshigher when the source contained glucose conjugates of quercetin. SeeHollman et al. FEBS Lett. 1997 Nov. 24; 418(1-2): 152-6.

Lung conditions may be treated by aerosol inhalation as this may bringthe treatment into direct contact with the affected tissue. The processof breathing delivers gases and inhaled materials directly to lugtissue, but the therapeutic effect of aerosolized therapies is dependentupon the dose deposited and its distribution. A drug or other treatmentmust be deposited past the oropharyngeal region to achieve therapeuticeffectiveness in the lungs. The location of aerosol deposition, centralor peripheral airways or alveolar, and the uniformity of distribution ofthe inhaled treatment may also play a role in the treatment'seffectiveness.

Effectiveness of therapy may be compromised if an aerosol is deliveredto a part of the lung devoid of the targeted disease or receptor. Forexample, autoradiographic studies have shown that receptors for the β₂agonist albuterol are not uniformly distributed throughout the lung.These receptors are present in high density in the airway epitheliumfrom the large bronchi to the terminal bronchioles. Airway smooth musclehas a lower β₂ receptor density, greater in the bronchioles thanbronchi. However, more than 90% of all β₂ receptors are located in thealveolar wall, a region where no smooth muscle exists and whosefunctional significance is unknown.

Inhaled anti-inflammatory therapy is probably most beneficial whenevenly distributed throughout the lung, since inflammatory cells, suchas eosinophils, lymphocytes, macrophages, and dendritic cells, arepresent throughout the airways and the alveolar tissue in asthma.

It is an object of the invention to provide improved treatment systemsand methods for disease states and conditions including lung conditions.

DISCLOSURE OF INVENTION/SUMMARY

I have discovered that plant stem cell extracts can be applied to humantissues to enhance regeneration, to prevent or reduce oxidative damage,to reduce inflammation, and to selectively kill cancer cells withrelative sparing of normal cells.

In embodiments, the invention includes a system for treating a tissue ofthe body where the system includes a plant stem cell and a deliverydevice. The delivery device may be configured to deliver the plant stemcell product to an affected area of a body. In some embodiments, thetissue may be an internal tissue, that is, a tissue other than skin.

The delivery device may be any of an aerosolizing device, an injectiondevice, a topical applicator, an eyedrop applicator, or an eardropapplicator. In some embodiments, the delivery device may be selectedfrom a group consisting of a syringe having a hollow needle, anaerosolizing device, an eyedrop applicator, a condom, a buccalapplicator, a suppository, an ingestible capsule, and an eardropapplicator.

An injection device may comprise a syringe and hollow needle sized forsubcutaneous injection, for intradermal injection, for intramuscularinjection, for intravenous injection, for intracapsular injection,intraarticular injection, for intraosseous injection, forintraperitoneal injection, for intracavernous injection, or for cardiacinjection.

In some embodiments, the invention includes a system for treating a lungcondition, where the system includes a plant stem cell product and anaerosolizing device. The device is configured to create an aerosol fromthe plant stem cell product and to deliver the aerosol to the lung viainhalation.

The plant stem cell may include one or more of a plant stem cellextract, a lyophilized plant stem cell, or an intact plant stem cell.The plant stem cell may also include an excipient, such as lactose,mannose, sodium chloride, a poly lactic acid, poly(lactic-co-glycolicacid), glycerol, a glycol, a surfactant, a plant gum such as xanthangum, or a liposome.

The plant stem cell product may be encapsulated.

The plant stem cell product may be derived from a dedifferentiated cellfrom any plant species. In embodiments, the a dedifferentiated cell maybe selected from a species in a group consisting of apple, lithy tree,mulberry, Cannabis, hemp, mint, grape, Eucalyptus, lingonberry,lungwort, oregano, plantain, poppy, elecampane, Lobelia, orchid, osharoot, garlic, ginger, turmeric, sage, mullein, licorice root, coltsfoot,thyme, Adhatoda vasica, Caraka samhita, Albizzia lebbeck, Boswelliaserrata, Curcuma longa, Ocimum sanctum, and Piper longum.

The dedifferentiated cell may include a stem cell recovered from a partof a plant regenerating from an injury or the progeny of such a stemcell. The part of a plant may include one or more of a stem, a meristem,a leaf, a flower, a fruit, a seed, a root, or a callus.

In embodiments, the plant stem cell product may include an apple stemcell product. The apple stem cell product may comprise an extract of anapple stem cell, where the apple stem cell is derived fromdedifferentiated cells recovered from an injured portion of an appleplant. The apple stem cell product may be encapsulated in a liposomecomprising a phospholipid, glycerin, and xanthan gum. In some of theseembodiments, the aerosolizing device includes an electronic-cigarette.

The plant stem cell stem cell may be from an Asparagales species or froma species that has in its telomeres the repeated 6-mer sequence TTAGGG.The telomere may include a plurality of contiguous copies of the TTAGGGsequence. The plant stem cell may be derived from a monocot Asparagalesspecies. In other embodiments, the plant stem cell may have in itstelomeres the repeated 12-mer sequence CTCGGTTATGGG (SEQ ID NO:1).

In other embodiments, the invention includes a plant SC product appliedin a divisible vehicle. A divisible vehicle is a composition that may beapplied to a part of the body so that a plant SC product may treatdirectly the part of the body or may be transported by the body to adifferent part of the body to be treated. Transport by the body mayinclude suspension, dissolution, or extraction in a body fluid such asblood, interstitial fluid, tears, saliva, phlegm, or mucous. Examples ofdivisible vehicles include creams, gels, polymers, lubricants, mixtures,emulsions, gums, or oils in which plant SC products may be suspended ordissolved. A plant SC product applied in a divisible vehicle may includeany of the combinations described above with respect to embodiments thatinclude a delivery device.

The aerosolizing device may include one or more of a nebulizer, apressurized metered-dose inhaler, a personal vaporizer, a humidifier, apersonal diffuser, a traditional cigarette, or an electronic-cigarette.

In embodiments, the invention includes a method of treating condition ata treatment site in a respiratory system of a mammal. The methodincludes steps of providing a plant stem cell product, aerosolizing theplant stem cell product, and delivering the aerosolized plant stem cellproduct for inhalation into the respiratory system. The aerosolizedplant stem cell may be delivered at a flow rate and with a particle sizedistribution such that at least 30% of the plant stem cell reaches atargeted treatment site. In other embodiments, the invention includes amore general method of treating a portion of the body by providing aplant stem product and a delivery device configured to deliver theproduct to the portion of the body to be treated, and delivering theplant stem cell product to the portion to be treated using the deliverydevice. In some embodiments, the tissue may be an internal tissue only,that is, a tissue other than skin. Some skin treatments may benefit froman appropriate delivery device. For example, a condom treated with aplant SC product may serve to as a delivery device the plant SC productto either male or female genitals.

The targeted treatment site for the method may include one or more of abronchus, a bronchiole, an alveolus, a lung parenchyma, or a capillarybed. The particle size distribution may include more than 50% ofparticles less than about 5 μm. In embodiments, the particle sizedistribution may include a range of about 0.5 μm to about 5 μm where thetreatment site includes a bronchus or a bronchiole. The particle sizedistribution may include a range of about 100 nm to about 600 nm wherethe treatment site includes an alveolus, a lung parenchyma, or acapillary bed.

The plant stem cell product used in the method may include any of thecombinations described above with respect to the system of theinvention. The plant stem cell product may include one or more of aplant stem cell extract, a lyophilized plant stem cell, a plant stemcell culture medium, or an intact plant stem cell. The plant stem cellmay also include a pharmacologically suitable excipient.

The treated lung condition may include one or more of a cancer, asthma,a lung inflammation, an infection, chronic obstructive pulmonarydisease, emphysema, cystic fibrosis, and a lung irritation. The methodmay treat the lung condition through one or more of lung regeneration, abronchodilatory effect, a tissue regeneration effect, a tumorsuppression effect, a cancer cytotoxic effect, a clearing of freeradicals, a cartilage regeneration effect, an anti-inflammatory effect,and a mucus reduction. In embodiments, the method treats a lungcondition through one or more of an antioxidant effect, ananti-inflammatory effect, a regeneration effect, or a cytotoxic effect.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows micrographs of selected results of scratch assays in lungepithelial cell cultures treated with an apple stem cell embodiment ofthe plant extracts of the invention.

FIG. 2 shows a graph of summary results of the scratch assays of FIG. 1.

FIG. 3A-3G show micrographs of scratch assays for cells treated withcontrols or embodiments of plant cell materials of the invention.

FIG. 4 is a summary graphical representation of control-ratioed scratchassay results exemplified in FIGS. 3A-G.

FIG. 5 shows a graph of TNF-α release inflammatory response fromstimulated mouse macrophages when treated with an apple stem cellembodiment of the invention.

FIG. 6 shows a graph of cytotoxicity activity of an apple stem cellembodiment of the invention on lung tumor cell lines A549 and NCIH520and on a lung epithelial cell line.

FIG. 7 shows fluorescence micrographs of acridine orange/ethidiumbromide staining of A549 cells treated with an apple stem cellembodiment of the invention.

FIG. 8 shows fluorescence micrographs of acridine orange/ethidiumbromide staining of NCI-H520 cells treated with an apple stem cellembodiment of the invention.

FIG. 9 shows fluorescence micrographs of Calcein AM staining in A549Lung Cancer Cell Line cells treated with an apple stem cell embodimentof the invention.

FIG. 10 shows fluorescence micrographs of Calcein AM staining inNCI-H520 Lung Cancer Cell Line cells treated with an apple stem cellembodiment of the plant stem cell of the invention.

FIG. 11 shows a graph of cytotoxicity activity of an apple stem cellembodiment of the invention on lung tumor cell lines A549 as measured bylactate dehydrogenase release.

FIG. 12 shows a graph of cytotoxicity activity of an apple stem cellembodiment of the invention on lung tumor cell line NCIH520 and on alung epithelial cell line as measured by lactate dehydrogenase release.

FIG. 13 shows a graph of reduction of oxidation produced by an applestem cell embodiment of the invention.

DETAILED DESCRIPTION

I have discovered that plant stem cells beneficially affect human (orother mammalian) tissues and cells. Exposure may be to plant stem cellsor to their products, contents, or enriched media (collectively “plantSC products”). As used herein, enriched media refers to culturematerials in which plant stem cells are grown and that is subsequentlyharvested. Such enriched media contains stem cell products, contents,and by-products of plant stem cell growth.

Human tissues are subject to injury from defects, diseases, insults,injury, trauma, or conditions including one or more of oxidative damage,inflammation, wounds, or cancer. The plant stem cell products of myinvention may treat such injuries by a combination of effects, includingantioxidant effects, anti-inflammatory effect, regeneration effects, orcytotoxic effects.

Oxidative damage can arise from normal metabolism, including aerobicrespiration such as mitochondrial electron transport. Oxidative damagemay also arise in response to infectious organisms or immune systemaction. Oxidative damage may also arise from therapeutic interventions,including radiation therapy. While cells and tissues possess endogenousprotective agents against oxidative damage, these agents may bedepleted. In some circumstances the endogenous protective agents, whichinclude reservoirs of small molecule antioxidants as well as inducibleantioxidant enzymes, may not be adequate to completely prevent oxidativeinjury. Notably, these endogenous protective agents diminish with age. Ihave found in a model system that plant SC products have antioxidantactivities that may prevent or reduce oxidative damage to affected cellswhen applied to the cells.

Inflammation is a response of the body to infection, irritation, ortissue damage. Inflammation marshals the body's defenses to effectivelyrespond to these conditions. In some circumstances, secondary effects ofinflammation produce additional harm. In other circumstances,inflammatory reactions may arise in inappropriate situations, such as inautoimmune diseases.

Inflammation is mediated by cellular communication, which may be throughcell-to-cell contact or through the release of soluble cytokines. Thesecytokines contribute to modulation of the response, to recruitment ofadditional cells to the inflammatory process, and to targeted attackagainst organisms or tissues recognized as foreign. Many cytokines areintimately associated with some of the undesirable effects ofinflammation. For example, Tumor necrosis factor alpha (TNF-α) is a cellsignaling protein (cytokine) involved in systemic inflammation and isone of the cytokines that make up the acute phase reaction.Specifically-targeted injectables, such as humanized monoclonalantibodies with specificity to TNF-α (e.g. adalimumab), can amelioratesome of the undesirable effects of inflammation in autoimmuneconditions.

I have found that plant SC products can act upon activated inflammatorycells in a model system to reduce the release of inflammatory cytokines,including TNF-α. The plant SC products may thus have a beneficial effecton inappropriate inflammatory activity when applied to the activatedcells. The plant SC products, because they prevent release of cytokines,may have a synergistic effect to agents such as adalimumab because ofthe different mechanisms of action, i.e. preventing TNF-α release asopposed to inactivating already-released TNF-α.

Wound healing is a beneficial response to insult or injury where cellsadjacent to the site of an injury or recruited to the site of an injuryrespond by closing wounds and repairing or replacing damaged tissue.

I have found that plant SC products can encourage tissue regenerationand wound healing in a model system. The plant SC products may have abeneficial effect on the regeneration of tissues following trauma orinjury when applied to the injured tissue.

Cancer is an abnormal replication and infiltration of cells that haveescaped regulatory control. It may be caused by accumulation ofmutations at many loci to produce a large variety of phenotypes.Recently developed drugs targeted to specific pathways have beeneffective against many forms of cancer, but the continuing accumulationof mutations frequently allows some tumor cells to escape this specifictherapy.

The fallback treatment in many cases remains cytotoxic agents that killtumor cells less discriminately. Such cytotoxic agents are valuable ifthey are more lethal to cancer cells than to the exposed normal cells. Atherapeutic ratio of greater than one indicates that a treatment killscancer cells more readily than noncancer cells. I have found that plantSC products, when applied to cells of a model system, can be more thanten times more effective at killing cancer cells than normal cells. Theplant SC products may have a beneficial effect if brought into contactwith cancer cells.

Somewhat paradoxically, plant SC products may protect cells fromoxidative damage, promote regeneration, and may have a cytotoxic effect.The net effects may depend on access to the treated cells, theinflammatory and oxidative environments, the treatment concentrationsand exposure times, or other factors.

Lung conditions may benefit from direct application of plant SC productsto lung tissues. I have found that a suitable method for providingexposure to plant SC products is aerosol delivery of plant SC productsto a treatment site in the respiratory system of a mammal.

Plant stem cells, such as apple stem cells, may stimulate human stemcells, protect against reactive oxygen species or uv-induced cell death,mitigate aging related alterations in gene expressions, and reverse orblock telomere shortening. Other plant stem cells such as those fromlithy tree, mulberry, Cannabis, hemp, mint, grape, Eucalyptus,lingonberry, lungwort, oregano, plantain, poppy, elecampane, Lobelia,chaparral (Larrea tridentate), orchid, osha root, garlic, ginger,turmeric, sage, mullein, licorice root, coltsfoot, thyme, Adhatodavasica, Caraka samhita, Albizzia lebbeck, Boswellia serrata, Curcumalonga, Ocimum sanctum, or Piper longum may have similar or otherbeneficial effects. In some cases, a particular plant stem cell may notbe suitable for some applications. For example, U.S. Pat. No.8,617,621B2 to Lim et al. described feeding of a dried ginseng stem cellpreparation to mice that increased the activity of mice NK cells. Thisis contrary to the observed anti-inflammatory effects of my preparation.Thus, stem cell products derived from ginseng stem cells may not besuitable for all applications. In other cases, plant stem cell extractsmay have an anti-inflammatory effect but may not support other effects.For example, KR101212032B1 to Lee et al. describe chrysanthemum-derivedstem cells as having an anti-inflammatory effect on a mouse macrophagecell line. No other effects were described; stem cell products derivedfrom chrysanthemum may not be suitable for all applications. In stillother cases, a plant stem cell may be from a plant that is known toproduce medications effective against certain cancers. For example, U.S.Pat. No. 8,790,927 B2 to Park, et al. described an anticancer effect inmice from ingestion of a cell line derived from a Taxus (yew) cambium.Taxus species are the original source of the chemotherapeutic drugpaclitaxel. While Park asserted that the cell line tested negative forpaclitaxel, he also reported that when the cell line was cultured in amedium containing an elicitor, it can produce paclitaxel at a highconcentration. It is thus possible that the cell line owes itsanticancer effects to the production of the known drug paclitaxel.Because this would be an effect of the drug rather than an effect of theplant stem cell product itself, Taxus-derived plant stem cell productsmay not be suitable for all applications. It is expressly within thescope of my invention to exclude certain stem cell types. In someembodiments, the systems and methods of my invention may be limited toplant stem cell products other than ginseng or Taxus stem cells.

Without intent to be bound by theory, Applicants believe that certainplant stem cells, because they share at least some signal motifs withother eukaryotes including humans, can produce beneficial modulation ofhuman signaling pathways. For example, Arabidopsis-type 7-mer stem celltelomerase may act on the repetitive human 6-mer telomere sequenceTTAGGG. Asparagales type 6-mer stem cell telomerase may also act on thehuman telomere with its identical sequence specificity. The longevolutionary distance between human and plant telomerase enzymessuggests that the plant enzymes may act in different circumstances andunder different conditions than the human enzyme. This likely differentaction may fortuitously remedy defects in human metabolism and cellproliferation, particularly when defects are related to cellproliferation or cellular senescence.

There may be other as yet unidentified convergences in plant and humansignaling pathways that present further opportunities for therapeuticeffects.

Further, since plant stem cells include genes and pathways that mayproduce biologically active compounds, these cells may be capable ofdelivering such active compounds to treat human disease.

As discussed above with respect to Ephedra, systemic application ofplant products may cause unwanted effects. Further, some plant cellcompounds may not be well absorbed systemically as discussed above forunconjugated quercetin. Most macromolecules cannot be administeredorally because proteins are digested before they are absorbed into thebloodstream. Also, their large size prevents them from naturally passingthrough the skin or nasal membrane, and therefore they may not beadministered transdermally without the use of penetration enhancers.

There remain a range of administration methods that can bring plant SCproducts into contact with tissue to be treated. These include ingestionin protected form, injection, transluminal arterial or venous delivery,topical application to mucous membranes of the mouth, nose, eyes,oropharynx, genitals, or digestive tract, or topical application to skinwith penetration enhancers. These administrative routes involvespecialized delivery devices.

In embodiments, the invention includes a plant SC product and a deliverydevice configured to deliver the plant SC product to a particularlocation of the body. This beneficially supports localized treatment toavoid untargeted effects. The delivery device may be any of anaerosolizing device, an injection device, a topical applicator, aneyedrop applicator, an eardrop applicator, a condom, a buccalapplicator, a rectal or vaginal suppository, an ingestible capsule, acatheter, or another device with a similar ability to deliver an aliquotof plant SC products to a targeted treatment site. In some embodiments,the tissue may be an internal tissue, that is, a tissue other than skin,because most parts of the skin may be directly accessible without adelivery device.

An injection device may comprise a syringe and hollow needle sized forsubcutaneous injection, for intradermal injection, for intramuscularinjection, for intravenous injection, for intracapsular injection,intraarticular injection, for intraosseous injection, forintraperitoneal injection, for intracavernous injection, or for cardiacinjection. These devices are well-known in the art and will not befurther described.

Catheters may be used as delivery devices to deliver plant SC productsto the gastrointestinal system or to the urethra or bladder.Angio-catheters may be used as delivery devices to deliver plant SCproducts to any of a large number of selected locations in thevasculature. For example, a catheter inserted transluminally into thefemoral or the brachial artery can reach most major blood vessels todeliver plant SC products to the vasculature of an organ or a localizedsegment of tissue. These devices are well-known in the art and will notbe further described. In other embodiments, a suppository containing aplant SC product may be inserted into the rectum or vagina to treattissue there. An ingestible capsule containing a plant SC product may beswallowed to treat portions of the digestive tract or to treat the bodysystemically.

Topical applicators exist in a variety of forms such as cotton orfoam-tipped swabs, adhesive patches, wound dressings, pipettes, andbandages. When skin is injured and the treatment is of the injurydirectly, the topical applicator may be directly applied to the injuredskin. As noted above, in some embodiments, the tissue may be an internaltissue only, that is, a tissue other than skin. Some skin treatments maybenefit from an appropriate delivery device. For example, a condomtreated with a plant SC product may serve to apply the plant SC productto either male or female genitals. The plant SC product may be mixedwith a lubricant applied to a condom.

When used to apply plant SC products to uninjured skin, a topicalapplicator may incorporate permeation enhancers. Permeation enhancersmay reversibly compromise the skin's barrier function and allow theentry of otherwise poorly penetrating materials. Permeation enhancersinclude materials such as fatty acids, terpenes, fatty alcohol,pyrrolidone, sulfoxides, laurocapram, surface active agents, amides,amines, lecithin, polyols, quaternary ammonium compounds, silicones, oralkanoates.

In some embodiments, the skin's barrier function may be degradedmechanically by delivery devices that abrade surface skin (dermabrasion)or pricking with sharp applicators such as microneedle arrays. In otherembodiments the skin's barrier function may be degraded electrically byapplying an electroporation voltage to the treated region.

Plant SC products in the form of eyedrops may be administered to thesclera of the eye using a pipette as the delivery device. Similarly,plant SC products in the form of eardrops may be administered into theear canal.

An aerosolizing device delivers finely divided portions such as dropletsfor inhalation. Aerosols containing plant SC products may be produced bya variety of devices including sonic nebulizers, e-cigarettes,vaporizers, powder or liquid droplet inhalers, humidifiers, or nasalsprays. The size of aerosol droplets, their concentration, and airflowvelocity profile determine the distribution of product delivery in thelung.

In other embodiments, the invention includes a plant SC product appliedin a divisible vehicle. A divisible vehicle is a composition that may beapplied to a part of the body so that a plant SC product may treatdirectly the part of the body or may be transported by the body to adifferent part of the body to be treated. Transport by the body mayinclude suspension, dissolution, or extraction in a body fluid such asblood, interstitial fluid, tears, saliva, phlegm, or mucous. Examples ofdivisible vehicles include creams, gels, polymers, lubricants, mixtures,emulsions, gums, or oils in which plant SC products may be suspended ordissolved. A plant SC product applied in a divisible vehicle may includeany of the combinations described in this application with respect toembodiments that include a delivery device.

A divisible vehicle such as a cream, lotion, gel, or emulsion containinga plant SC product may be applied to the skin, to mucosa, to the scalp,or to a wound. A polymer mixture such a chewing gum containing a plantSC product may be used to expose the buccal mucosa to the plant SCproduct for an extended period. Saliva developed while chewing the gummay transport the plant SC products to the buccal mucosa. A mixture suchas a mouthwash containing a plant SC product may be applied to the oralcavity for an extended period, thereby treating portions of the oralmucosa. A surfactant or conditioner mixture such as a shampoo orconditioner containing a plant SC product may be applied to treat thehair or scalp. A lubricant mixture containing a plant SC product mayused during coitus to deliver the plant SC product to the genitals.

In some embodiments, the plant SC products include an excipient.Suitable excipients include one or more of lactose, mannose, sodiumchloride, poly lactic acid, poly (lactic-co-glycolic acid), glycerol,glycol, a surfactant, or a liposome. The excipient may serve to bufferthe plant SC products or to adjust its viscosity or heat capacity totune the aerosol generation process. Excipients may also adjusthydrophilicity of the materials to control aerosol particle size.

Plant SC products may be treated by encapsulation to help enhancestability by protecting the plant SC products from exposure toenvironmental materials or conditions that may degrade the activity ofthe active materials. Encapsulation may also serve to control therelease of active materials to a desired time (e.g. when exposed to thelung surface) or at a desired rate. Encapsulation may be performed byany method known in the art, including those reviewed by Yadav et al. inPeptides 32 pp. 173-187 (2011). This review is hereby incorporated byreference for its disclosure of methods of encapsulation. Any of theseencapsulation methods may be used with any of the delivery devices forany appropriate treatment site.

In some embodiments, a suitable method of encapsulation includesemulsification polymerization using aqueous phase methacrylate monomerand a photoinitiator such as benzoin ethyl ether emulsified with plantSC products with polyethylene oxide as a stabilizer and exposure to UVlight after emulsification to produce poly(methacrylate) encapsulatedactive components of plant SC products. The capsules may range fromabout 50 to about 5000 nm in diameter. While the capsules may be closeto monodisperse (depending on the method of preparation), in someembodiments, the size of capsules may be deliberately widely distributedto control the rate of release of active materials. Widely distributedpopulations of capsules may be prepared by altering the conditions ofemulsification during encapsulation or by mixing two or more batches ofcapsules with different size.

When intact plant stem cells are used, any capsules must be as large orlarger than the size of the cells. When plant stem cell contents areextracted or removed from the intact cells then the capsule size is nolonger limited by the cellular size. Such capsules may be selectedprimarily based upon the targeted region of the lung or upon sizes thatmay be more compatible with aerosolization processes. For example, ifplant stem cell contents are removed by lysis, homogenization, orultrasonic disruption, or when plant SC products include an enrichedmedium that is subsequently harvested (and optionally further purifiedor concentrated), these removed contents may be encapsulated.

In other embodiments, encapsulated plant SC products may be prepared asphospholipid nano-emulsions or as nano-liposomes.

Capsules containing plant SC products may be washed by dialysis, bycentrifugal filtration, by tangential flow filtration, by centrifugationand decanting, or by other techniques known in the art, to producewashed encapsulated plant SC products. Washing helps remove unreactedmonomers or initiator as well as materials not incorporated in capsules.Alternatively, and depending on the materials used in the encapsulationprocess, encapsulated plant SC products may be used without furtherprocessing. After washing, encapsulated plant SC products may beresuspended in a buffer, in sterile saline, in water, or in a suspensioncontaining other excipient materials. In some embodiments, theresuspension material may have viscosity, heat capacity, orhydrophilicity selected to optimize aerosolization of the plant SCproducts.

The location of deposition of aerosolized particles depends on aerosolsize distribution, sometimes expressed as mass median aerodynamicdiameter (MMAD). Fine aerosols are distributed in peripheral airways butdeposit less material per unit surface area than larger particleaerosols because their volume is lower. Larger particle aerosols depositmore drug per unit surface area, but this is preferentially targeted tolarger airways. The precise location of deposition depends on airwaycaliber and structure, which differ between individuals. In general,large conducting airways and oropharyngeal region receive aerosols witha MMAD of 5-10 μm. Smaller particles (1-5 μm in diameter) deposit insmall airways and alveoli. More than half of 3 μm particles deposit inthe alveolar region.

Individual pathology differences can also affect aerosol deposition. Forexample, the airway narrowing in mild to moderate asthma is moreresponsive to 2.8 μm aerosols than to either 1.5 μm or 5 μm aerosols.This is likely due to a combination of penetration depth anddistribution of affected tissue since smooth muscle (the tissue thatproduces airway narrowing) is not present in the alveolar region.

The size of particles in the lung is not necessarily the same as whenintroduced because the lung has a relative humidity of about 99.5%. Ahygroscopic aerosol delivered at relatively low temperature and humidityinto one of high humidity and temperature would increase in size duringinhalation. This effect is more important for smaller particles becauseof their higher surface area relative to volume. Suitable excipientssuch as salts or sugars may control water absorption for a morepredictable aerosol size distribution.

Particles not deposited during inhalation are exhaled and thus lost.Deposition due to sedimentation affects particles down to 0.5 μm indiameter, whereas below 0.5 μm, the main mechanism for deposition is bydiffusion.

The distribution of deposition of aerosolized particles also depends onthe position of the patient. For example, in experiments with 4micrometer aerosols, N R Labris and M B Dolovich in Br J Clin Pharmacol,56, 588-599 reported a 2:1 ratio between lower and upper lobes when thetreated person was upright. This gradient is reduced when the patient issupine.

Inhalable aerosols may be produced in a variety of methods includingswirl nozzles, venturi atomizers, T-jets, vibrating-mesh nebulizers,heated wicks, vibrating nozzles, and electrospray systems, among others.Most involve the interaction of a gas stream (usually air) and a liquidflow to break up the liquid into discrete particles separated by theflowing air. Suitable methods for delivering the plant SC products maybe categorized by the source of the gas stream and by how the liquidflow is broken up.

For inhaled aerosols, the gas stream may be produced by a pump or storedgas or by breathing. Pump or stored gas-based methods advantageouslyoffer repeatable flow rates and pressures and can reduce the work a userneed perform to inhale an aerosol. This reduction of work may be ofconsequence when the treated patient has reduced lung function that maybe a consequence of the treated condition.

In some embodiments, the invention delivers the plant SC products usinga nebulizer or a pressurized metered-dose inhaler. Nebulizers andpressurized metered-dose inhalers use pumps and stored gas toadvantageously produce aerosols that may be less dependent on usertechnique.

Nebulizers are common medical devices that use air pumps to produce (orhelp propel) aerosols for therapy. These convert liquids or suspensionsinto aerosols with a particle size that can be inhaled into the lowerrespiratory tract. There are pneumatic jet nebulizers, ultrasonicnebulizers, and mesh nebulizers. Some nebulizer designs may bebreath-enhanced or breath-actuated. These devices are well-known in theart and will not be further described.

Medical nebulizers advantageously permit adjustment of flow rates and(indirectly) aerosol size distribution. This permits more accuratetargeting of delivery of plant SC products to the desired treatmentlocation.

The pressurized metered-dose inhaler is a commonly-used device that usesstored gas under pressure for aerosol production and delivery. There arepress-and-breathe and breath-actuated pressurized inhaler designs. Thesedevices are well-known in the art and will not be further described.

Pressurized metered-dose inhalers advantageously are portable and easilyoperated. These devices typically are designed for use with relativelyhigh concentration medications (such as albuterol for asthma) so thataerosol volume per actuation is usually small. In some embodiments, theinvention includes use of pressurized metered-dose inhaler loaded withplant SC products and an inert drive gas. In such embodiments, the plantSC products may be concentrated to provide an effective dose in one or afew operations.

In other embodiments, the invention delivers plant SC products using adevice where the gas stream is produced by breathing. Such systemsadvantageously allow for aerosol delivery over an extended period oftime without interfering significantly with other life activities. Thismay especially valuable when treatment aerosols comprise fairly lowconcentration materials. Many plant SC product treatment compositionsmay be of fairly low concentration.

Breathing-based aerosol delivery devices include powder inhalers,traditional cigarettes, personal vaporizers, also known as personaldiffusers, electronic cigarettes, or e-cigarettes. Powder inhalers, suchas commercial Spinhaler and Twisthaler devices, deliver a dose of apowder during inhalation. Such devices may be used in embodiments of theinvention by drying the plant SC products to produce a finely dividedpowder. Traditional cigarettes combust materials to produce a smoke thatmay contain uncombusted components. These may be used in embodiments ofthe invention by applying the plant SC products to a combustiblematerial such as paper or leaves. Some personal vaporizers use heat toproduce an inhalable aerosol from a liquid or suspension drawn into awick by capillary action. Users inhale these aerosols and can controlpuff pressure, puff length, and interval between puffs. With somedevices, current applied to the heater may be adjusted. Other personalvaporizers, such as personal diffusers, may use heat to directly producea vapor that may then be inhaled.

Typical aerosols from personal vaporizers have particle diameters in the250-450 nm range and particle density concentration of approximately 109particles/cm³. These relatively small aerosol particles should target auser's entire lung, including the alveolar region. Because they may beused for extended period, a user may alter posture between upright andsupine to better distribute the aerosols to a targeted region.

In other designs, piezo-electric, ferroelectric, or magnetostrictivevibrators may produce the aerosol. Use of personal vaporizers includingvibration to produce aerosols may advantageously produce an unheatedaerosol that may be adjusted for particles in the micrometer range. Thismay be particularly appropriate for plant SC products that includeintact cells.

Although the variations in user operation parameters such as puffpressure can affect the rate of plant SC product delivery, use ofpersonal vaporizers can compensate for these variations by loading apredispensed dose of plant SC product into the personal vaporizer. Thedevice may then be used until the preloaded dose is exhausted. This maybe especially advantageous where extended treatment times are desired,as may be the case with plant SC products.

Personal vaporizers may be designed (or adjusted) to produce acontrolled aerosol particle size by adjusting the size of the capillaryopenings in the heated wick, by adjusting the current to the heater, orby adjusting the viscosity and heat capacity of excipients mixed withthe plant SC materials. The inventive method may include suchadjustments to control the aerosol size distribution. In embodiments,the size may be adjusted to produce aerosols having a size distributionin the range of about 100 nm to about 600 nm where the treatment siteincludes an alveolus, a lung parenchyma, or a capillary bed. Where thetreatment site includes a bronchus or a bronchiole, the particle sizedistribution may be adjusted to include particles in a range of about0.5 μm to about 5 μm.

Many personal vaporizers include breath sensors that may permitadjustment of heater current during inhalation. In some embodiments, theinvention includes tuning of heater current to produce a desired aerosolsize distribution. This may include individual adjustment based uponbreath sensor measurement.

The examples below describe a collection of experiments designed todemonstrate the effectiveness of plant stem cell products in modelsystems comprising cultured cell lines including human lung cell linesand mouse macrophage cell lines. Scratch assays used a normal humanepithelial line to ascertain the effect of plant stems on cellregeneration. A cytokine release assay evaluated anti-inflammatoryactivity using an activated mouse macrophage cell line. Two human lungcancer cell lines (a lung adenocarcinoma and a squamous carcinoma) werecompared to a normal lung epithelial cell line to determine treatmentefficacy and cancer-specific cytotoxicity.

Apple stem cell extracts were tested in each of the assay types. Otherplant stem cells, as well as other plant extracts, were evaluated inscratch assays using the normal human epithelial line and in cytoxicityassays comparing effects on the human lung adenocarcinoma cell line andthe normal human epithelial cell line.

The purpose of testing plant extracts other than plant SC products wasto provide a comparison. Plant extracts other than plant SC productswould be expected to have some effects in some cases. Such extracts arethe source of many types of effective drugs, so some effects arereasonably expected.

Example 1: Plant Stem Cell Extracts

The following plant stem cell extracts were tested for biologicaleffects. Plant stem cells refer to dedifferentiated replicating cellsisolated from a part of a plant regenerating from an injury and theirprogeny. Injury sites may be from any portion of a plant such as callus,leaves, fruit, stems, flowers, roots, meristem, root cap, or seeds.Other plant stem cells may be derived from cells of a developing plantembryo, from a plant callus, or from plant tissue samples (explants) intissue culture medium in vitro. Plants stem cells may be derived frommany different cell types and may be able to differentiate into a wholeplant.

Apple stem cell culture extract (ASC) was purchased from LotioncrafterLLC of Eastsound, Wash. This composition included a lysate ofdedifferentiated cells from Malus domestica callus. The cells werederived from Swiss apple variety Uttwiler Spatlauber. The extract wasprepared by encapsulating a lysate of cultured cells in liposomescomposed of soy phospholipids (0.14% weight/volume (“w/v”)), glycerin(0.4% w/v), and xanthan gum (1% w/v). The liposomes were suspended inabout a 10% suspension in deionized water adjusted to pH 7.4 with 1.4%w/v phenoxyethanol as a preservative.

Lingonberry Stem Cell (LSC) extract was purchased fromMakingCosmetics.com Inc. of Snoqaulmie, Wash. This composition includedwater, glycerin, Vaccinium vitis idaea fruit extract, xanthan gum,sodium benzoate, gluconolactone, and calcium gluconate. Vaccinium vitisidaea is a short evergreen shrub in the heath family that bears ediblefruit, native to boreal forest and Arctic tundra throughout the NorthernHemisphere from Eurasia to North America.

Orchid Stem Cells (OSC) extract was purchased from MakingCosmetics.comInc. of Snoqaulmie, Wash. This composition included water, glycerin,Calanthe discolor extract, xanthan gum, sodium benzoate, gluconolactone,calcium gluconate. Calanthe discolor is a species of orchid native toeastern Asia.

Example 2: Plant Extracts

The following plant extracts other than plant stem cell extracts weretested for comparison with plant stem cell extracts.

Apple Fiber Powder (AFP) was purchased from Starwest Botanicals ofSacramento, Calif. This composition included powder from Pyrus malus.Pyrus malus is a former taxonomic grouping applied to the apples, pearsand related plants of the subfamily Maloideae.

Dandelion Root Extract (DRE) was purchased from Starwest Botanicals ofSacramento, Calif. This composition included Taraxcum officinale rootextract, water, and alcohol (30%). Taraxcum officinale is a floweringherbaceous perennial plant of the family Asteraceae (Compositae). It canbe found growing in temperate regions of the world.

Aloe Vera Juice (AVJ) was purchased from Starwest Botanicals ofSacramento, Calif. This composition included decolorized Aloebarbedensis, citric acid and sodium benzoate. Aloe barbedensis is asucculent plant species of the genus Aloe. An evergreen perennial, itoriginates from the Arabian Peninsula but grows wild in tropicalclimates around the world and is cultivated for agricultural andmedicinal uses.

Ginkgo Leaf Extract (GLE) extract was purchased from Starwest Botanicalsof Sacramento, Calif. This composition included Ginkgo biloba Leafextract, water and alcohol (30%). Ginkgo biloba is a large tree nativeto China; the tree is widely cultivated.

Example 3: Target Cells

Human lung adenocarcinoma cell line A549, human squamous carcinoma cellsline NCI-H520, and “normal” lung epithelial cell line L132 were procuredfrom National Centre for Cell Sciences (NCCS), Pune, India. RAW 264.7mouse macrophage cell lines were used for inflammation assays.

Example 4: Scratch Gap Regeneration Assay I

Scratch assay determines effects of a treatment on cell migration andproliferation. In a typical scratch assay, a “scratch or wound gap” iscreated in monolayer cell culture by scratching and creating a gap inthe culture. “Healing” of the gap by growth and cell migration towardsthe center of the gap is monitored and measured. Various factors thatalter the migration and growth of the cells to bridge the gap can leadto increased or decreased “healing” rate of the gap. Scratch assay onnormal lung cell line L132 was performed to evaluate regenerativepotential of the apple stem cell extract.

Method: Human lung epithelial cell line L132 cells were cultured inDulbecco Modified Eagle Medium with 10% fetal bovine serum (FBS). Cellswere seeded (0.05×10⁶) into 24-well tissue culture plate. At about 80%confluence, a scratch in a straight line was created across the centerof the well with a sterile 1 ml pipette tip. The long axial of the tipwas held perpendicular to the bottom of the well to create a uniformscratch. Wells were washed after the scratch and then supplemented withfresh culture media. Test wells were subjected to the media with thetest material at one of 100 and 250 μg per mL of media. Media withoutadded test materials served as a control. Cells were then cultured foranother 24 hours, washed twice with PBS and then fixed with 3.7%Paraformaldehyde for 30 minutes. Pictures of the monolayer were taken ona microscope and the gaps were quantitatively evaluated using ImageJsoftware from (http://rsb.info.nih.gov/ij/download.html). All studieswere performed in triplicate. Results of triplicate data points appearin the table below. Concentrations of ASC refer to a mass per volume ofthe lysate.

TABLE 1 Results of ASC scratch assay as analyzed by ImageJ software.Values are determined widths of scratched areas. replicate 1 replicate 2replicate 3 ASC Scratch Assay initial gap width (mm) Average SD SEControl 27.32 28.90 25.55 27.26 1.68 0.97 100 μg/mL 24.43 25.55 26.6525.54 1.11 0.64 250 μg/mL 24.42 25.55 25.55 25.17 0.65 0.38 replicate 2ASC Scratch Assay replicate 1 final gap width (mm) replicate 3 AverageSD SE Control 21.21 20.23 20.21 20.55 0.57 0.33 100 μg/mL 12.21 13.3213.33 12.95 0.64 0.37 250 μg/mL 10.21 11.12 10.90 10.74 0.47 0.27

FIG. 1 shows the physical appearance of scratches in selected wells forthis ASC scratch assay. The parallel lines disposed roughly verticallyin each image are the boundaries of the scratch as determined by theImageJ software.

difference in width: 0-24 standard hours replicate 1 replicate 2replicate 3 mean deviation Control 6.11 8.67 5.34 6.71 1.42 100 μg/ml12.22 12.23 13.32 12.59 0.52 250 μg/ml 14.21 14.43 14.65 14.43 0.18Ratio/control 100 μg/ml 2.00 1.41 2.49 1.88 0.22 250 μg/ml 2.33 1.662.74 2.15 0.21

Table 2 shows results calculated from the data of from Table 1 whereeach value was calculated as the difference in gap width between timezero and 24 hours, with replicates compared individually to remove gapwidth influence on results.

standard Ratio/control mean deviation 100 μg/ml 1.88 0.22 250 μg/ml 2.150.21

Table 3 shows results calculated from the data of from Table 1 whereeach value was calculated by forming a ratio of the mean difference ofTable 2 to the mean difference of the control wells. Standard deviationswere calculated by error propagation assuming errors were uncorrelated.These summary results showed a higher degree of scratch closure for thetreated wells, with a higher degree of closure at the higher ASCconcentration.

FIG. 2 is a graphical representation of these ASC scratch assay results.This shows a clear acceleration in closure of the gap in the treatmentgroup as compared to untreated controls. The apple stem cell extractsproduced faster regeneration of the cells in the gap at both theconcentrations used after 24 hours treatment. The rate of gap closurewas treatment dose dependent. Results were statistically significant atp≤0.05, indicating that apple stem cell extract can exert a positiveeffect in wound closing and regeneration of lung tissue.

Example 5: Scratch Gap Regeneration Assay II

The experiment of Example 4 was repeated, substituting other plantmaterials for ASC. Plant stem cell materials included Apple Fiber Powder(AFP), Dandelion Root Extract (DRE), Aloe Vera Juice (AVJ), Ginkgo LeafExtract (GLE), Lingonberry Stem Cells (LSC), Orchid Stem Cells (OSC) asdescribed in Examples 1 and 2. Example 5 experiments were performed witha common set of control wells. Concentrations of each material refer tomass of each as-supplied material. Each material was first prepared as a1000 μg/mL stock in in Dulbecco Modified Eagle Medium with 10% FBS.

FIGS. 3A-3G shows monochrome images of phase contrast micrographs ofselected wells of control and treated cells at the start of theexperiment and 24 hours later (fixed with paraformaldehyde). The linesrunning roughly parallel to one another in each micrograph are theboundaries of the scratched region as determined by the ImageJ software.FIG. 3A shows control-treated wells. FIGS. 3B through 3G show one wellfor each treatment at each concentration at the start of the experimentand the same well 24 hours later. Note the increased number of cells inthe scratched gap after 24 hours.

TABLE 4 Results of Control (unaugmented media, AFP, DRE, AVJ, GLE, LSC,and OSC scratch assays as analyzed by ImageJ software. Values are thedetermined widths in mm of scratched areas at t = 0 and at 24 hours.Entries in rows marked diff. are differences between width of scratchedareas at t = 0 and at 24 hours for the same well. replicate 1 replicate2 replicate 3 Average SD SE Control t = 0 28.90 29.98 29.87 29.58 0.590.34 Control t = 24 27.89 27.75 27.65 27.76 0.12 0.07 Control diff. 1.012.23 2.22 1.82 0.57 0.33 AFP, t = 0 100 μg/mL 29.21 29.09 28.9 29.070.16 0.09 250 μg/mL 28.9 29.78 29.88 29.52 0.54 0.31 AFP, t = 24 100μg/mL 26.78 25.65 25.54 25.99 0.69 0.4 250 μg/mL 25.67 24.32 25.67 25.220.78 0.45 AFP diff. 100 μg/mL 2.43 3.44 3.36 3.08 0.46 0.26 250 μg/mL3.23 5.46 4.21 4.30 0.91 0.53 DRE, t = 0 100 μg/mL 28.92 28.78 28.7728.82 0.08 0.05 250 μg/mL 28.9 29 28.76 28.89 0.12 0.07 DRE, t = 24 100μg/mL 26.21 26.55 27.21 26.66 0.51 0.29 250 μg/mL 26.22 25.43 25.44 25.70.45 0.26 DRE diff. 100 μg/mL 2.71 2.23 1.56 2.17 0.47 0.27 250 μg/mL2.68 3.57 3.32 3.19 0.37 0.22 AVJ, t = 0 100 μg/mL 28.97 29.29 28.7829.18 0.53 0.31 250 μg/mL 28.79 28.09 28.94 28.61 0.45 0.26 AVJ, t = 24100 μg/mL 25.67 26.77 26.45 26.3 0.57 0.33 250 μg/mL 23.67 23.21 28.9425.27 3.18 1.84 AVJ diff. 100 μg/mL 3.3 2.52 2.33 2.72 0.42 0.24 250μg/mL 5.12 4.88 0 3.33 2.36 1.36 GLE, t = 0 100 μg/mL 28.88 29.9 28.9729.53 0.56 0.33 250 μg/mL 29.09 29.78 28.9 29.26 0.46 0.27 GLE, t = 24100 μg/mL 27.89 26.98 26.56 27.14 0.68 0.39 250 μg/mL 24.56 23.45 24.8724.29 0.75 0.43 GLE diff. 100 μg/mL 0.99 2.92 2.41 2.11 0.82 0.47 250μg/mL 4.53 6.33 4.03 4.96 0.99 0.57 LSC, t = 0 100 μg/mL 28.78 29.8729.56 29.4 0.56 0.32 250 μg/mL 28.77 29.77 29.78 29.44 0.58 0.34 LSC, t= 24 100 μg/mL 27.65 27.16 27.89 27.57 0.37 0.21 250 μg/mL 24.32 24.3325.21 24.62 0.51 0.30 LSC diff. 100 μg/mL 1.13 2.71 1.67 1.84 0.66 0.38250 μg/mL 4.45 5.44 4.57 4.82 0.44 0.25 OSC, t = 0 100 μg/mL 28.9 28.7829 28.89 0.11 0.06 250 μg/mL 28.97 28.98 28.78 28.91 0.11 0.07 OSC, t =24 100 μg/mL 25.43 26.21 24.43 25.36 0.89 0.52 250 μg/mL 24.32 24.4423.21 23.99 0.68 0.39 OSC diff. 100 μg/mL 3.47 2.57 4.57 3.54 0.82 0.47250 μg/mL 4.65 4.54 5.57 4.92 0.46 0.27

Treatment Mean ratio standard deviation AFP 100 μg/mL 1.69 0.35 250μg/mL 2.36 0.38 DRE 100 μg/mL 1.19 0.38 250 μg/mL 1.75 0.34 AVJ 100μg/mL 1.49 0.35 250 μg/mL 1.83 0.77 GLE 100 μg/mL 1.16 0.50 250 μg/mL2.73 0.37 LSC 100 μg/mL 1.01 0.48 250 μg/mL 2.65 0.33 OSC 100 μg/mL 1.940.39 250 μg/mL 2.70 0.33

Table 5 shows results calculated from the data of from Table 4 whereeach value was calculated by forming a ratio of the mean differences ofTable 4 to the mean differences of the control wells. Standarddeviations were calculated by error propagation assuming errors wereuncorrelated. These summary results showed a higher degree of scratchclosure for the treated wells, with a higher degree of closure at thehigher plant material concentrations. The number of replicates may notbe sufficient to show reasonable statistical significance for theeffects in all cases. AFP, GLE, LSC, and OSC had the most outstandingperformance in promoting regeneration under the conditions of thisassay. Each of the plant stem cell extracts outperformed all but one ofthe non-stem-cell materials.

FIG. 4 is a summary graphical representation of the control-ratioedscratch assay results (including the ASC results from Example 4). Notethe consistent values where scratch results for treated cells aregreater than one, particularly at the higher tested concentrations, withthe higher concentration plant stem cell extracts consistently promotingregeneration in this assay.

Example 6: TNF-α Cytokine Release Assay

In an inflammatory reaction, activated cells (such as macrophages)release a variety of pro-inflammatory cytokines (such as tumor necrosisfactor alpha (TNF-α). The released cytokines can be assayed as a measureof inflammatory activity. To evaluate the anti-inflammatory role ofapple stem cell extracts, mouse RAW 264.7 cell lines mouse macrophageswere used as an adherent monolayer on petri dishes. These cells could beharvested easily without damage caused by enzymes or cell scrapers. Themacrophages were stimulated in suspension with lipopolysaccharide (LPS)to initiate an inflammatory response. Cells were seeded into 12-wellcell culture plates containing the apple stem cell extract treatmentmaterials. After 16-18 hours, the medium conditioned by the macrophageswas harvested and the cytokine profile in the medium determined withenzyme-linked immunosorbent assays (ELISA) by measuring TNF-α levels.

Method: Three concentration of ASC (6.25, 12.5 and 25 μg/mL in media)were tested for the anti-inflammatory effect. RAW 264.7 mouse macrophagecells were maintained in DMEM containing Glutamax supplemented with 10%FBS, penicillin (100 U/ml) and streptomycin (100 μg/ml). The macrophagestreated with LPS (1:500 dilution of a 0.1 mg/ml solution of LPS inphosphate buffered saline (PBS)) to produce a pro-inflammatory response.The ASC treatment was performed with a final concentration of 1×10⁵macrophages in wells of a 12-well plate. The cytokine assay wasperformed using a TNF-α ELISA from R&D Systems of Minneapolis, Minn.

Results indicated (Table 6, FIG. 5) that LPS alone produced aninflammatory response more than 1000 times that of unstimulated cells asmeasured by TNF-α expression. Treatment with ASC on the inducedmacrophages showed a dose-dependent decrease of TNF-α expression. ASCconcentrations of 6.25, 12.5, and 25 μg/mL reduced TNF-α activity in theinduced cells by 72.1, 92.1 and 94.5%, respectively. This reduced TNF-αat doses of 12.5 and 25 μg/ml was statistically significant with p≤0.05for 25 μg/ml and p≤0.02 in 12.5 μg/ml. The apple stem cell extracts thusexerted an anti-inflammatory effect on the activated macrophage cells.

TABLE 6 Apple Stem Cell percent inhibition Extract Conc. (82 g/ml)TNF-α(μg/ml) vs. LPS 25 481.89 94.5 12.5 687.9 92.1 6.25 2432.89 72.1LPS 8712.63 0 unstimulated 6.45 Results of TNF-α release assay showinganti-inflammatory effects of apple stem cell extracts on of three mouseRAW 264.7 macrophage cell line cells. Values shown are averages sets ofexperiments. ASC extracts dramatically reduced inflammatory responses inthe target cells, as exemplified by reduced TNF-α release (greaterinhibition of inflammation).

Example 7: MTT Cell Proliferation Assay I

The MTT Cell Proliferation assay determines cell survival followingapple stem cell extract treatment. The purpose was to evaluate thepotential anti-tumor activity of apple stem cell extracts as well as toevaluate the dose-dependent cell cytotoxicity.

Principle: Treated cells are exposed to3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). MTTenters living cells and passes into the mitochondria where it is reducedby mitochondrial succinate dehydrogenase to an insoluble, colored (darkpurple) formazan product. The cells are then solubilized with DMSO andthe released, solubilized formazan is measured spectrophotometrically.The MTT assay measures cell viability based on the generation ofreducing equivalents. Reduction of MTT only occurs in metabolicallyactive cells, so the level of activity is a measure of the viability ofthe cells. The percentage cell viability is calculated against untreatedcells.

Method: A549 and NCI-H520 lung cancer cell lines and L132 lungepithelial cell line were used to determine the plant stem celltreatment tumor-specific cytotoxicity. The cell lines were maintained inMinimal Essential Media supplemented with 10% FBS, penicillin (100 U/ml)and streptomycin (100 μg/ml) in a 5% CO₂ at 37 Celsius. Cells wereseeded at 5×10³ cells/well in 96-well plates and incubated for 48 hours.Triplicates of eight concentrations of the apple stem cell extract wereadded to the media and cells were incubated for 24 hours. This wasfollowed by removal of media and subsequent washing with the phosphatesaline solution. Cell proliferation was measured using the MTT CellProliferation Kit I (Boehringer Mannheim, Indianapolis, Ind.). Newmedium containing 50 μl of MTT solution (5 mg/ml) was added to each welland cultures were incubated a further 4 hours. Following thisincubation, DMSO was added and the cell viability was determined by theabsorbance at 570 nm by a microplate reader.

In order to determine the effectiveness of apple stem cell extracts asan anti-tumor biological agent, an MTT assay was carried out and IC50values were calculated. IC50 is the half maximal inhibitory functionconcentration of a drug or compound required to inhibit a biologicalprocess. The measured process is cell death.

Results: ASC-treated Human lung adenocarcinoma cell line A549.

TABLE 7 Results of cytotoxicity of apple stem cell extract on lungcancer cell line A549 as measured by MTT assay (performed intriplicate). Values of replicates are % of cell death. Results:ASC-treated Human squamous carcinoma cell line NCI-H520. Concentration*replicate replicate replicate Mean of % Live (μg/ml) 1 2 3 replicates SDSEM Cells 250 93.18 90.86 90.34 91.46 1.51 0.87 8.54 100 86.88 85.1885.69 85.92 0.87 0.50 14.08 50 80.58 79.49 81.04 80.37 0.80 0.46 19.6325 74.28 73.81 76.39 74.83 1.38 0.79 25.17 12.5 67.98 68.13 71.75 69.282.13 1.23 30.72 6.25 61.67 62.45 67.10 63.74 2.93 1.69 36.26 3.125 55.3756.77 62.45 58.20 3.75 2.16 41.80 1.562 49.07 51.08 57.80 52.65 4.572.64 47.35 0.781 42.77 45.40 53.15 47.11 5.40 3.12 52.89

TABLE 8 Results of cytotoxicity of apple stem cell extract on lungcancer cell line NCI-H520 as measured by MTT assay (performed intriplicate). Values of replicates are % of cell death. Results:ASC-treated lung epithelial cell line L132. Concentration* replicatereplicate replicate Mean of % Live (μg/ml) 1 2 3 replicates SD SEM cell250 88.28 89.29 87.73 88.43 0.79 0.46 11.57 100 78.13 79.19 78.13 78.480.61 0.35 21.52 50 67.98 69.09 68.54 68.54 0.56 0.32 31.46 25 57.8358.99 58.94 58.59 0.66 0.38 41.41 12.5 47.68 48.89 49.34 48.64 0.86 0.5051.36 6.25 37.53 38.79 39.75 38.69 1.11 0.64 61.31 3.125 27.37 28.6930.15 28.74 1.39 0.80 71.26 1.562 17.22 18.59 20.56 18.79 1.68 0.9781.21 0.781 7.07 8.48 10.96 8.84 1.97 1.14 91.16

TABLE 9 Results of cytotoxicity of apple stem cell extract on lungepithelial cell line L132 as measured by MTT assay (performed intriplicate). Values of replicates are % of cell death. Summary Results:cytotoxicity of apple stem cell extracts. Concentration* replicatereplicate replicate Mean of % Live (μg/ml) 1 2 3 replicates SD SEM cell250 39.51 42.52 44.03 42.02 2.30 1.33 57.98 100 32.93 34.44 33.69 33.690.75 0.44 66.31 50 30.60 28.94 30.52 30.02 0.94 0.54 69.98 25 27.9627.81 27.13 27.63 0.44 0.25 72.37 12.5 25.62 25.55 25.40 25.52 0.12 0.0774.48 6.25 23.13 20.87 18.61 20.87 2.26 1.31 79.13 3.125 13.34 11.0811.83 12.08 1.15 0.66 87.92 1.562 6.56 7.31 9.57 7.81 1.57 0.91 92.190.781 8.06 4.30 3.54 5.30 2.42 1.40 94.70

TABLE 10 Target Cell Line IC50 A549 12.58 NCl-H520 10.21 L132 127.46IC50 values of the apple stem cell extracts on the on the target celllines as determined by MU assay.

Apple stem cell extracts killed lung cancer cells lines A549 andNCI-H520 at relatively low doses: IC50s were 12.58 and 10.21 μg/mlrespectively as compared to 127.46 μg/ml for the lung epithelial cellline L132. Near complete anti-tumor activity was seen at a dose of 250μg/ml in both the lung cancer cell lines. This same dose spared morethan one half of the L132 cells. See Tables 7-10. The data revealed thatapple stem cell extract is cytotoxic to lung cancer cells while sparinglung epithelial cells. FIG. 6 shows a graphical representation ofcytotoxicity activity of apple stem cell extracts on lung tumor celllines A549, NCIH520 and on L132 lung epithelial cell line (marked“Normal”). The Y-axis is the mean % of cells killed by the indicatedtreatment compared to unexposed cells. The difference in cytotoxicitylevels was statistically significant at p≤0.05.

Example 9: MTT Cell Proliferation Assay II

The experiment of Example 7 was repeated substituting other plantmaterials for ASC. Plant stem cell materials included Dandelion RootExtract (DRE), Aloe Vera Juice (AVJ), Apple Fiber Powder (AFP), GinkgoLeaf Extract (GLE), Lingonberry Stem Cells (LSC), Orchid Stem Cells(OSC) as described in Examples 1 and 2. The concentrations of plantmaterials used were nominally 250, 100, 50, 25, 6.25, 3.125, 1.562, and0.781 μg/mL. These materials were tested only for cells the human lungepithelial cell line L132 (as a proxy for normal epithelial cells) andfor cells of the human lung adenocarcinoma cell line A549 (as a proxyfor lung cancer cells).

A549 cells lung cancer cell line cytotoxicity results for each of thetreatment materials.

DRE-treated lung cancer cell line A549 cells.

TABLE 11 Triplicate results of cell death of DRE-treated A549 cellsmeasured by MTT assay. Percentage of live cells calculated as 100%-Meanof triplicates. AVJ-treated lung cancer cell line A549 cells.Concentration % (μg/mL)-DRE-treated Live A549 % of cell death Mean SDSEM cell 250 80.43 76.40 74.84 77.23 2.89 1.67 22.77 100 67.60 75.2663.77 68.88 5.85 3.38 31.12 50 65.32 62.94 59.94 62.73 2.70 1.56 37.2725 56.83 57.97 48.14 54.31 5.38 3.11 45.69 6.25 55.59 49.69 49.17 51.483.57 2.06 48.52 3.125 51.76 48.45 45.34 48.52 3.21 1.85 51.48 1.56243.69 44.00 36.02 41.24 4.52 2.61 58.76 0.781 37.47 26.19 19.57 27.749.05 5.23 72.26

TABLE 12 Triplicate results of cell death of AVJ-treated A549 cellsmeasured by MTT assay. Percentage of live cells calculated as 100%-Meanof triplicates. AFP-treated lung cancer cell line A549 cells.Concentration % (μg/mL)-AVJ-treated Live A549 % of cell death Mean SDSEM cell 250 76.81 78.16 75.88 76.95 1.14 0.66 23.05 100 76.40 75.2673.71 75.12 1.35 0.78 24.88 50 65.32 66.15 59.94 63.80 3.37 1.95 36.2025 50.10 48.45 56.63 51.73 4.32 2.50 48.27 6.25 47.52 46.38 46.17 46.690.72 0.42 53.31 3.125 39.86 38.61 43.79 40.75 2.70 1.56 59.25 1.56232.40 19.77 30.54 27.57 6.82 3.94 72.43 0.781 20.50 15.63 32.19 22.778.51 4.92 77.23

TABLE 13 Triplicate results of cell death of AFP-treated A549 cellsmeasured by MTT assay. Percentage of live cells calculated as 100%-Meanof triplicates. GLE-treated lung cancer cell line A549 cells.Concentration % (μg/mL)-AFP-treated Live A549 % of cell death Mean SDSEM cell 250 86.13 87.99 86.65 86.92 0.96 0.56 13.08 100 79.50 81.0682.09 80.88 1.30 0.75 19.12 50 73.60 72.46 71.33 72.46 1.14 0.66 27.5425 68.01 67.70 66.98 67.56 0.53 0.31 32.44 6.25 60.87 62.11 60.77 61.250.75 0.43 38.75 3.125 49.48 51.76 50.72 50.66 1.14 0.66 49.34 1.56240.06 41.72 47.00 42.93 3.62 2.09 57.07 0.781 39.23 37.78 36.85 37.961.20 0.69 62.04

TABLE 14 Triplicate results of cell death of GLE-treated A549 cellsmeasured by MTT assay. Percentage of live cells calculated as 100%-Meanof triplicates. LSC-treated lung cancer cell line A549 cells.Concentration % (μg/mL)-GLE-treated Live A549 % of cell death Mean SDSEM cell 250 88.42 91.49 90.44 90.12 1.56 0.90  9.88 100 84.39 83.7783.16 83.77 0.61 0.35 16.23 50 79.47 81.58 76.75 79.27 2.42 1.40 20.7325 73.60 72.54 71.40 72.51 1.10 0.63 27.49 6.25 62.89 63.68 59.91 62.161.99 1.15 37.84 3.125 50.18 54.47 51.84 52.16 2.17 1.25 47.84 1.56246.93 44.30 43.33 44.85 1.86 1.07 55.15 0.781 39.56 39.39 40.96 39.970.87 0.50 60.03

TABLE 15 Triplicate results of cell death of LSC-treated A549 cellsmeasured by MTT assay. Percentage of live cells calculated as 100%-Meanof triplicates. OSC-treated lung cancer cell line A549 cells.Concentration (μg/mL) % Live LSC treated A549 % of cell death Mean SDSEM cell 250 77.54 78.85 78.20 78.20 0.65 0.38 21.80 100 77.14 76.0476.59 76.59 0.55 0.32 23.41 50 66.42 68.52 66.82 67.25 1.12 0.65 32.7525 59.80 67.22 64.16 63.73 3.73 2.15 36.27 6.25 50.53 48.82 48.07 49.141.26 0.73 50.86 3.125 41.14 43.60 42.72 42.49 1.24 0.72 57.51 1.56239.47 39.74 40.61 39.94 0.60 0.34 60.06 0.781 38.55 31.83 36.79 35.723.48 2.01 64.28

TABLE 16 Triplicate results of cell death of OSC-treated A549 cellsmeasured by MTT assay. Percentage of live cells calculated as 100%-Meanof triplicates. L132 cells (“normal” lung epithelial cell line)cytotoxicity results for each of the treatment materials. DRE-treatedlung epithelial cell line L132 cells. Concentration (μg/mL) % LiveOSC-treated A549 % of cell death Mean SD SEM cell 250 70.84 65.57 71.4969.30 3.25 1.87 30.70 100 48.81 50.91 57.28 52.33 4.41 2.55 47.67 5046.59 49.60 53.33 49.84 3.38 1.95 50.16 25 38.77 40.81 36.58 38.72 2.111.22 61.28 6.25 35.74 40.79 41.05 39.19 3.00 1.73 60.81 3.125 34.5533.68 37.02 35.08 1.73 1.00 64.92 1.562 33.86 33.44 27.63 31.64 3.482.01 68.36 0.781 21.32 20.00 34.82 25.38 8.21 4.74 74.62

TABLE 17 Triplicate results of cell death of DRE-treated L132 cellsmeasured by MTT assay. Percentage of live cells calculated as 100%-Meanof triplicates. AVJ-treated lung epithelial cell line L132 cells.Concentration (μg/mL) % DRE-treated Live L132 % of cell death Mean SDSEM cell 250 86.66 86.61 86.66 86.64 0.03 0.02 13.36 100 76.29 77.3976.84 76.84 0.55 0.32 23.16 50 65.92 68.17 67.01 67.03 1.13 0.65 32.9725 55.54 58.95 57.19 57.23 1.70 0.98 42.77 6.25 45.17 49.73 47.37 47.422.28 1.32 52.58 3.125 34.80 40.50 37.54 37.61 2.85 1.65 62.39 1.56224.42 31.28 27.72 27.81 3.43 1.98 72.19 0.781 14.05 22.06 17.89 18.004.01 2.31 82.00

TABLE 18 Triplicate results of cell death of AVJ-treated L132 cellsmeasured by MTT assay. Percentage of live cells calculated as 100%-Meanof triplicates AFP-treated lung epithelial cell line L132 cells.AFP-treated lung epithelial cell line L132 cells. Concentration (μg/mL)AVJ-treated % Live L132 % of cell death Mean SD SEM cell 250 57.03 55.9353.62 55.53 1.74 1.00 44.47 100 50.99 49.78 47.04 49.27 2.03 1.17 50.7350 44.95 43.63 40.45 43.01 2.31 1.34 56.99 25 38.91 37.49 33.86 36.752.60 1.50 63.25 6.25 32.88 31.34 27.28 30.50 2.89 1.67 69.50 3.125 26.8425.19 20.69 24.24 3.18 1.84 75.76 1.562 20.80 19.05 14.11 17.98 3.472.00 82.02 0.781 14.76 12.90 7.52 11.73 3.76 2.17 88.27

TABLE 19 Triplicate results of cell death of AFP-treated L132 cellsmeasured by MTT assay. Percentage of live cells calculated as 100%-Meanof triplicates AFP-treated lung epithelial cell line L132 cells.GLE-treated lung epithelial cell line L132 cells. Concentration (μg/mL)% AFP-treated Live L132 % of cell death Mean SD SEM cell 250 56.15 55.4357.19 56.26 0.88 0.51 43.74 100 49.95 48.24 47.64 48.61 1.20 0.69 51.3950 43.74 41.05 38.09 40.96 2.83 1.63 59.04 25 37.54 33.86 28.54 33.324.53 2.61 66.68 6.25 31.34 26.67 18.99 25.67 6.24 3.60 74.33 3.125 25.1419.48  9.44 18.02 7.95 4.59 81.98 1.562 18.94 12.29 10.87 14.03 4.312.49 85.97 0.781 12.73  5.10  6.81  8.21 4.00 2.31 91.79

TABLE 20 Triplicate results of cell death of GLE-treated L132 cellsmeasured by MTT assay. Percentage of live cells calculated as 100%-Meanof triplicates AFP-treated lung epithelial cell line L132 cells.LSC-treated lung epithelial cell line L132 cells. Concentration (μg/mL)GLE-treated % Live L132 % of cell death Mean SD SEM cell 250 84.42 83.2083.08 83.57 0.74 0.43 16.43 100 80.05 79.29 78.59 79.31 0.73 0.42 20.6950 72.75 71.59 74.10 72.81 1.26 0.72 27.19 25 80.05 81.86 79.99 80.631.06 0.61 19.37 6.25 68.26 70.13 68.26 68.88 1.08 0.62 31.12 3.125 60.6263.07 60.62 61.44 1.41 0.82 38.56 1.562 48.07 48.77 48.83 48.56 0.420.24 51.44 0.781 46.27 45.57 46.67 46.17 0.56 0.32 53.83

TABLE 21 Triplicate results of cell death of LSC-treated L132 cellsmeasured by MTT assay. Percentage of live cells calculated as 100%-Meanof triplicates AFP-treated lung epithelial cell line L132 cells.OSC-treated lung epithelial cell line L132 cells. Concentration (μg/mL)LSC-treated % Live L132 % of cell death Mean SD SEM cell 250 86.41 85.8285.76 86.00 0.35 0.20 14.00 100 81.21 81.27 79.99 80.82 0.72 0.42 19.1850 75.96 74.74 73.51 74.74 1.23 0.71 25.26 25 74.74 72.75 71.47 72.991.65 0.95 27.01 6.25 70.13 68.32 68.26 68.90 1.06 0.61 31.10 3.125 54.0358.05 53.44 55.17 2.51 1.45 44.83 1.562 53.97 51.98 51.98 52.64 1.150.66 47.36 0.781 46.79 45.62 44.92 45.78 0.94 0.54 54.22

TABLE 22 Triplicate results of cell death of OSC-treated L132 cellsmeasured by MTT assay. Percentage of live cells calculated as 100%-Meanof triplicates AFP-treated lung epithelial cell line L132 cells.Calculated values. Concentration (μg/mL) OSC-treated % Live L132 % ofcell death Mean SD SEM cell 250 61.84 62.37 60.44 61.55 1.00 0.57 38.45100 54.14 53.44 52.10 53.23 1.04 0.60 46.77 50 42.94 42.30 40.32 41.851.37 0.79 58.15 25 35.94 34.48 33.31 34.58 1.32 0.76 65.42 6.25 33.9632.67 32.03 32.89 0.98 0.57 67.11 3.125 27.48 26.20 26.72 26.80 0.650.37 73.20 1.562  9.80  7.29  7.35  8.15 1.43 0.83 91.85 0.781  7.29 8.98  8.05  8.11 0.85 0.49 91.89

TABLE 23 Calculated IC50 doses (ug/mL) and therapeutic ratios (IC50 forL132 cells/IC50 for A549 cells) for each treatment material. Valuesgreater than one indicate that a material would be more selective inkilling cancer cells than normal cells. ASC results imported fromExample 8. ASC DRE AVJ AFP GLE LSC OSC A549 12.58 9.822 11.48 11.98 11.113.7 33.9 IC50 L132 IC50 127.46 56.88 62.66 82.65 77.636 9.267 15.38Ther. 10.1 5.8 5.5 6.9 7.0 0.7 0.5 Ratio

These studies indicate that at least some of the materials may beeffective anti-cancer agents. While some stem cell products arenon-selective in this assay, ASC has outstanding selectivity compared toother materials.

Example 10: Staining of Cells

Apoptosis is a programmed cell death that eliminates physiologicallyredundant, physically damaged, and abnormal cells. Various stainingprocedures can help elucidate the mechanisms of cytotoxic effects, andin particular whether cytotoxic effects are attributable to apoptosis ascompared to purely physical effects.

Ethidium bromide and acridine orange are used to visualize the cellapoptosis in a cell culture upon treatment with a biological agent or adrug. Acridine orange is a vital dye and will stain both live and deadcells. Ethidium bromide will stain only cells that have lost membraneintegrity. Live cells will appear uniformly green. Early apoptotic cellswill stain green and contain bright green dots in the nuclei as aconsequence of chromatin condensation and nuclear fragmentation. Lateapoptotic cells will also incorporate ethidium bromide and thereforestain orange, but, in contrast to necrotic cells, the late apoptoticcells will show condensed and often fragmented nuclei. Necrotic cellsstain orange, but have a nuclear morphology resembling that of viablecells, with no condensed chromatin.

Calcein AM staining helps confirm cell viability. Calcein AM is anon-fluorescent, hydrophobic compound that easily permeates intact, livecells. In live cells the nonfluorescent calcein AM is converted to agreen-fluorescent calcein after acetoxymethyl ester hydrolysis byintracellular esterases.

Method: A549 and NCI-H520 lung cancer cell lines were cultured in DMEMsupplemented with 10% FBS, 4 mM L-glutamine, 1% penicillin/streptomycinunder a fully humidified atmosphere containing 5% CO₂ at 37 Celsius. Forexperiments, cells were collected from sub confluent monolayers bytrypsinization with trypsin/EDTA. Cell viability was determined usingtrypan blue dye exclusion staining. In all experiments. Apple stem cellextracts (6.25, 12.5, and 25 μg/mL) and 0.1% dimethyl sulfoxide vehiclecontrols were sterilized with UV and placed in wells of 6 well plates.Culture medium containing A549 or NCI-H520 cells were added andincubated according to the protocols below.

Calcein AM staining: The control and treated cells (1×10⁵ per well) wereincubated with the apple stem cell extracts for 24 hours at 37 Celsiusand 5% CO₂. The culture media was aspirated off followed by cell washingwith ice cold PBS. 2 μM Calcein-AM and was added and cells incubated for10 min at 37 Celsius. Cells were examined under a fluorescencemicroscope provided with a triple filter set (excitation: 400, 495, 570nm; emission: 460, 530, 610 nm), and combined with digital camera (CanonPowerShot G8). Viability was expressed as percentage cells retainingcalcein (green fluorescence) compared to the total cells counted.

Acridine orange/ethidium bromide (AO/EtBr) Staining: The control andtreated cells (3×10⁴ per well) were incubated with the apple stem cellextracts for 48 hours at 37 Celsius and 5% CO₂. After incubation, cellswere fixed in methanol: glacial acetic acid (3:1) for 30 min at roomtemperature, washed with PBS and stained with 1:1 ratio of AO/EtBr.Stained cells were immediately washed with PBS and viewed under afluorescence microscope (Nikon, Eclipse TS100, Japan) with amagnification of ×40.

The number of cells expressing apoptotic features was counted andexpressed as a fraction of the total number of cells present in thefield.

Results: FIGS. 7-8 show selected fields of treated cells stained withAO/EtBr. Each field has the associated apple stem cell extract treatmentdose indicated in the corner. Note that magnification changes betweenimages to better indicate the relative number of cells. Normal tumorcells, early and late apoptotic cells, and necrotic cells were examinedusing fluorescent microscopy. Early-stage apoptotic cells were marked bycrescent-shaped or granular yellow-green acridine orange nuclearstaining. Late-stage apoptotic cells were marked with concentrated andasymmetrically localized orange nuclear ethidium bromide staining.Necrotic cells increased in volume and showed uneven orange-redfluorescence at their periphery. Cells appeared to be in the process ofdisintegrating.

The percentage of apoptotic lung cancer cells of both cell linesdetected by AO/EtBr staining was significant at 12.5 μg/mL of treatmentdose of apple stem cell extract.

FIGS. 9-10 show selected fields of treated cells stained with CalceinAM. Each field has the associated apple stem cell extract treatment doseindicated in the corner. Note that magnification changes between imagesto better indicate the relative number of cells.

Consistent with the AO/EtBr staining, a dose of 12.5 μg/mL wasassociated with significantly fewer tumor cells exhibiting the greenfluorescence indicative of viable target cells when treated with applestem cell extract and stained with Calcein-AM. Response to the applestem cell extracts was dose-dependent; more viable target cells werepresent in wells treated with lower doses of apple stem cell extracts.

These AO/EtBr and Calcein-AM staining results support that treatmentwith apple stem cell extracts produces apoptosis and bio-sensitivity inlung tumor cell lines.

Example 11: Lactate Dehydrogenase (LDH) Release Assay

LDH is released upon tumor cell death. Measuring released LDH canconfirm cytotoxic effects. LDH is a stable cytosolic enzyme that isreleased from the cell upon cell lysis. The LDH assay is based onquantitatively measuring released LDH using a coupled enzymatic assay.Released LDH converts a tetrazolium salt into a red soluble formazanproduct which then can be measured colorimetrically. The amount of LDHreleased is proportional to the number of lysed cells.

Method: A549 and NCI-H520 cells were cultured as described in Example 5and treated with varying concentrations of apple stem cell extract(0.781, 1.562, 3.125.6.25.12.5, 25, 50, 100, and 250 μg/mL) ofExample 1. The cells were treated at 37 Celsius for 45-60 min, then thesupernatant containing released LDH was harvested and transferred into afresh 96-well plate. 50 μL of substrate mix was added to each wellcontaining the transferred supernatant. The plate was incubated for 30min at room temperature and the reaction stopped by adding 50 μL of stopsolution to each well. Absorbance of the solutions was measured at 490nm in a plate reader and the results expressed as a percentage of LDHreleased (n=4±S.D.) compared to maximum LDH released from lysed controlcells.

Results: Significant released LDH activity was observed with doses of100 and 250 μg/ml correlating to about 78% cell cytotoxicity in both thelung cancer cell lines (FIG. 11 for A549 cells and FIG. 12 for NCI-H520cells). The data are consistent with the MTT assay results showinganti-tumor cytotoxic action of apple stem cell extract treatments.

Example 12: Antioxidant Activity

The human body both produces and is subject to free radicals. Recoveryof injury to any body tissue is frequently manifested by antioxidantenzyme levels. Superoxide anions are produced by dedicated signalingenzymes and as a byproduct of metabolism (e.g. mitochondrialrespiration). Inhaled pathogens can also induce airway cells to producethese and other reactive oxidant species (ROS). For example, inhalationof the ubiquitous environmental fungus Aspergillus fumigatus canexacerbate airway inflammation. Inhaled ROS and those endogenouslyformed by inflammatory cells constitute an increased intrapulmonaryoxidative burden. Despite their potential toxicity, superoxide ion andsome of its derivatives, especially hydrogen peroxide (H₂O₂), are alsosignaling molecules that mediate a variety of biological responses suchas cell proliferation, differentiation, and migration. The enzymesinvolved in repair of free radical-induced DNA damage may be especiallyimportant in preventing cancerous transformation.

When produced in excess, free radicals and oxidants generate oxidativestress, a deleterious process that can seriously alter cell membranesand other structures such as proteins, lipids, lipoproteins, anddeoxyribonucleic acid. Free radicals, especially superoxide, andnon-radicals, such as H₂O₂, can be generated in quantities large enoughto overwhelm endogenous protective enzyme systems, such as superoxidedismutase (SOD), catalase (CAT), and reduced glutathione. The balancebetween oxidants and antioxidants (known as redox balance) is altered inmany diseases with severe consequences. The pathophysiologicalmechanisms by which free radicals generate various types of stress(oxidative stress, nitrative, carbonyl, inflammatory, endoplasmicreticulum stress etc.) culminate in diseases such as chronic obstructivepulmonary disease, bronchial asthma, bronchiectasis, and idiopathicpulmonary fibrosis. Studies have shown that there is induction of SODand CAT in inflamed tissues generally and in inflammatory lung diseases.

The endogenous antioxidant defense system is essential to maintain redoxbalance. Human tissue produces enzymes that protect against freeradicals and ROS. The SOD enzyme helps convert superoxides to H₂O₂. H₂O₂may be metabolized by other enzymes such as CAT and glutathioneperoxidase. Overexpression of CAT has been associated with impairedpost-ischemic neovascularization; this response of elevated anti-oxidantenzymes, though necessary to combat oxidative stress, has detrimentalsecondary consequences to tissue recovery and repair.

Measuring the antioxidant activity of an exemplary treatment extract ofthe invention illustrates whether the extract can exert a protectiveantioxidant effect. The assays below provide strongly oxidizingenvironments though addition or generation of an oxidizer (a superoxideradical in the SOD assay and H₂0₂ in the CAT assay). Addition ofdifferent concentrations of apple stem cell extract (see Example 1) mayneutralize or inactivate part or all of the oxidizing material. Residual(unneutralized) oxidizer in the assays react with other reagents in themixtures to provide a measurable absorbance proportional to the amountof remaining oxidizer. The loss of absorbing material (compared to acontrol with no ASC added) is a measure of the antioxidant activity ofthe ASC. Values close to zero show low antioxidant activity. Valuesclose to 100% show full neutralization of the oxidizer and hence astronger antioxidant activity. Higher values indicate a protectiveantioxidant potential of the plant stem cell extract.

These assays are labeled SOD and CAT because they measure antioxidantactivity associated with these enzymes, if present. The assay results donot speak to the antioxidation mechanism. The ASC extract may includeactive antioxidant enzymes, other antioxidants, such as ascorbate ortocopherol, or combinations of these and other antioxidant materials.

Method: The SOD assay generates a superoxide radical of riboflavin thatreacts with hydroxylamine hydrochloride to form nitrite. The nitritereacts with sulphanilic acid in a Griess reagent to produce a diazoniumcompound which subsequently reacts with naphthylamine to produce a redazo compound with absorbance measured at 543 nm. In the assay, 100 μLaliquots of diluted ASC extract in a range of concentrations were eachcombined with 1.4 mL of an assay buffer (50 mM Phosphate buffer, pH 7.4,20 mM L-Methionine, 1% (v/v) Triton X-100, 10 mM hydroxylamine, and 50mM EDTA) and 80 μL of a stock riboflavin solution. Absorbance oftriplicates were measured at 543 nm after addition of the Griessreagent.

CAT is a ubiquitous antioxidant enzyme that degrades hydrogen peroxideinto water and oxygen. The CAT assay method is based on the principlethat dichromate in acetic acid is reduced to chromic acetate when heatedin the presence of H₂0₂. Hydrogen peroxide concentration is directlyproportional to the concentration of chromic acetate produced asmeasured by absorbance at 610 nm. Antioxidants in differentconcentrations of ASC extracts in the assay preparation was allowed todegrade H₂0₂ for a fixed period. The reaction was stopped by theaddition of dichromate/acetic acid mixture and the remaining H₂O₂ wasdetermined by measuring chromic acetate colorimetrically after heatingthe reaction.

Results: superoxide radical inactivation in apple stem cell extracts atvarious concentrations. The assay was performed in triplicate, but thefirst replicate in each series was discarded due to an apparentsystematic error. The remaining replicates for each concentration wereaveraged and the difference in average absorbance between eachconcentration and the control (no ASC) were divided by the absorbance ofthe control tubes. The final values, expressed as a percentage, indicatethe fraction of the total superoxide radical deactivated.

The mean percent inactivation of superoxide radical as compared to ano-ASC control is presented Table 24 and in FIG. 13. The inactivationmonotonically increased with increasing concentrations of ASC. Atconcentrations of 50, 100 and 250 μg/mL ASC, the effect essentiallysaturated, probably limited by the assay conditions as evidenced by thevery steep slopes at low ASC concentration. The data suggest that theASC can protect against oxidative damage due to superoxide radicals.

TABLE 24 superoxide radical inactivation ASC concentration Mean %(μg/mL) (duplicates) 250 0.087 100 0.083 50 0.083 25 0.128 12.5 0.1456.25 0.169 3.125 0.175 1.562 0.186 0.871 0.203 Oxidation protection ofASC treated cells as measured by SOD assay. Reported values are means ofduplicates ratioed to control (no ASC).

Results: H₂0₂ inactivation. Peroxide inactivation was also measured inapple stem cell extracts at various concentrations. The assay wasperformed in triplicate to determine the antioxidant potential of applestem cell extracts at various concentrations. The inactivationmonotonically increased with increasing concentrations of ASC. Atconcentrations of 50, 100, and 250 μg/mL ASC, the anti-peroxide effectflattened, probably limited by the assay conditions as evidenced by thevery steep slopes at low ASC concentration. The data suggest that theASC can protect against oxidative damage due to H₂0₂. The mean percentinactivation of H₂0₂ is presented in Table 25 and in FIG. 13.

TABLE 25 H₂0₂ inactivation ASC concentration mean % (μg/mL)(triplicates) 250 85% 100 81% 50 75% 25 67% 12.5 60% 6.25 57% 3.125 48%1.562 31% 0.781 12% Oxidation protection of ASC treated cells asmeasured by CAT assay. Reported values are means of triplicates ratioedto control (no ASC).

The results show apple stem cell extracts can inactivate superoxideradicals and H₂0₂. This antioxidant activity suggests the materials mayhave value in treatment of pathologies including lung pathologiesarising from or exacerbated by reactive oxygen species.

Plant SC products have thus been shown to be effective agents formultiple effects. There is variation in the efficacy between stem celltypes and effects. ASC are more effective at selective cytotoxicity oflung cancer cells and LST and OSC are more effective in scratch assayrecovery. Some of these differences may be attributable to concentrationeffects, but others may be attributable to a different mix of materialspresent in the different cell types. Non-stem cell extracts have someeffects also, but the experiments indicate that the strong beneficialeffects are associated with plant SC products.

The embodiments described herein are referred in the specification as“one embodiment,” “an embodiment,” “an example embodiment,” etc. Thesereferences indicate that the embodiment(s) described can include aparticular feature, structure, or characteristic, but every embodimentdoes not necessarily include every described feature, structure, orcharacteristic. Further, when a particular feature, structure, orcharacteristic is described in connection with an embodiment, suchfeature, structure, or characteristic in may also be used in connectionwith other embodiments whether or not explicitly described. Further,where specific examples are given, the skilled practitioner mayunderstand the particular examples as providing particular benefits suchthat the invention as illustratively disclosed herein suitably may bepracticed in the absence of any element which is not specificallydisclosed herein or within that particular example.

This disclosure mentions certain other documents incorporated byreference. Where such documents conflict with the express disclosure ofthis document, this document shall control.

It will be apparent to those of ordinary skill in the art that manymodifications and variations of the described embodiment are possible inthe light of the above teachings without departing from the principlesand concepts of the disclosure as set forth in the claims.

Although the present disclosure describes certain exemplary embodiments,it is to be understood that such disclosure is purely illustrative andis not to be interpreted as limiting. Consequently, without departingfrom the spirit and scope of the disclosure, various alterations,modifications, and/or alternative applications of the disclosure will,no doubt, be suggested to those skilled in the art after having read thepreceding disclosure. Accordingly, it is intended that the followingclaims be interpreted as encompassing all alterations, modifications, oralternative applications as fall within the true spirit and scope of thedisclosure.

We claim:
 1. A system for treating a lung condition, the systemcomprising: a plant stem cell product, comprising a lingonberry stemcell product or an orchid stem cell product; and an aerosolizing deviceconfigured to create an aerosol from the plant stem cell product and todeliver the aerosol to the lungs via inhalation.
 2. The system of claim1, wherein the lingonberry stem cell product includes one or more of alingonberry stem cell extract, a lyophilized lingonberry stem cell, alingonberry stem cell enriched medium, or an intact lingonberry stemcell.
 3. The system of claim 2, wherein the lingonberry stem cell isderived from a dedifferentiated cell of a lingonberry plant.
 4. Thesystem of claim 3, wherein the dedifferentiated cell is derived from astem, a meristem, a leaf, a flower, a fruit, a seed, a root, a root cap,an embryo, an explant, or a callus of the lingonberry plant.
 5. Thesystem of claim 1, wherein the orchid stem cell product includes one ormore of an orchid stem cell extract, a lyophilized orchid stem cell, anorchid stem cell enriched medium, or an intact orchid stem cell.
 6. Thesystem of claim 5, wherein the orchid stem cell is derived from adedifferentiated cell of an orchid plant.
 7. The system of claim 6,wherein the dedifferentiated cell is derived from a stem, a meristem, aleaf, a flower, a fruit, a seed, a root, a root cap, an embryo, anexplant, or a callus of the orchid plant.
 8. The system of claim 1,wherein the plant stem cell product is encapsulated.
 9. The system ofclaim 8, wherein the plant stem cell product comprises an extractprepared by encapsulating a lysate of a cultured plant stem cell in aliposome.
 10. The system of claim 9, further comprising an excipient.11. The system of claim 10, wherein the excipient includes lactose,mannose, sodium chloride, a phospholipid, a poly lactic acid, apoly(lactic-co-glycolic acid), glycerol, a glycol, a surfactant, or axanthan gum.
 12. The system of claim 9, wherein the plant stem cellproduct is a lingonberry stem cell extract including glycerin,gluconolactone, and xanthan gum.
 13. The system of claim 9, wherein theplant stem cell product is an orchid stem cell extract includingglycerin, gluconolactone, and xanthan gum.
 14. The system of claim 1,wherein the aerosolizing device is a nebulizer, pressurized metered-doseinhaler, a personal vaporizer, a humidifier, a powder inhaler, apersonal diffuser, a traditional cigarette, or an electronic-cigarette.15. A system for treating a lung condition, the system comprising: aplant stem cell product, comprising a plant stem cell other thanginseng, yew, or chrysanthemum, the plant stem cell encapsulated in aliposome comprising a phospholipid, glycerin, and xanthan gum; and anaerosolizing device configured to create an aerosol from the plant stemcell product and to deliver the aerosol to the lungs via inhalation. 16.The system of claim 15, wherein the plant stem cell product comprises anapple stem cell product, a lingonberry stem cell product, or an orchidstem cell product.
 17. The system of claim 15, wherein the aerosolizingdevice is a nebulizer, pressurized metered-dose inhaler, a personalvaporizer, a humidifier, a powder inhaler, a personal diffuser, atraditional cigarette, or an electronic-cigarette.